Metallothioneins (MTs) are an superfamily of low-molecular-weight, cysteine (Cys)-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). However the mechanism that MTs regulate ROS is not still completely clear. Therefore in this work the activities of catalase (CAT) and superoxide dismutase (SOD) as two main antioxidant enzymes in Escherichia coli were studied in the presence of different plant MT isoforms. Previously four rice (Oryza sativa) MT isoforms OsMTI, OsMTI-2a, OsMTI-3a and OsMTII-1a that belong to type 1, type 2, type 3 and type 4, respectively, were heterologously expressed in Escherichia coli as carboxyl-terminal extensions of glutathione-S-transferase (GST). The four strains expressing these 4 isoforms were grown in LB medium containing 1- 3 mM hydrogen peroxide or 4-6 % ethanol. The tolerance of cells expressing recombinant proteins were compared to control strain (The strain that produce GST) by plotting their growth curve. The results showed that the tolerance of bacteria expressing MT is considerably more than that in control strain suggesting the role of heterologous expression of plant MTs in tolerance of bacteria against oxidative stress. The activities of catalase and SOD in the strains expressing MTs as well as control strain were determined. The enzyme assay showed that the activities of both CAT and SOD in strains expressing MTs were higher than that in control strain. However the activity of CAT differs in different strains which may indicate that different MT isoforms have different effects on the activity of CAT. In addition, In vitro study of the effect of MT isoforms on CAT and SOD activities were analyzed. To this end the recombinant Zn 2+ -MT and Fe 2+ -MT were expressed by addition of either Zn 2+ or Fe 2+ 20 min after addition of IPTG to the transgenic strains. The soluble proteins were extracted 4h after addition of IPTG and were purified using affinity chromatography. Then the pure MTs were added to total protein which was extracted from control strain and then the activities of CAT and SOD were analyzed. The results show that while the activity of SOD was increased in the presence of Zn 2+- MT, the activity of CAT was not changed. However the activity of CAT increased in the presence of Fe 2+ -MT. Therefore the results suggest that MTs play important role as supplier of Fe 2+ and Zn 2+ for CAT and SOD, respectively.
